Niemann Pick Type C (NPC1) is a rare but lethal pediatric dementia caused by a mutation in NPC1, a housekeeping protein residing in the late endosomal compartment with a putative role in cholesterol transport. The result is a severe lipidosis characterized by massive accumulation of lysosomal sterols and other lipids that ultimately cause cell death. The disease is of enormous basic science interest as well as a hallmark model of dysfunctional intracellular cholesterol trafficking and because of its similarities with Alzheimer's disease (AD), which suggest shared underlying mechanisms. To date strategies to model NPC have not yet determined how some mutations of NPC1 can cause neuronal failure in humans for two reasons: i) use of animal models that do not replicate all aspects of human pathology, and ii) a focus on accumulation of cholesterol as the cause of neuronal dysfunction in NPC, which may not be the predominant phenotype in neuronal cell populations. We propose to generate the first human neuronal model of NPC1 by genetic engineering of human embryonic stem cells (hESCs) and reprogramming of somatic cells into human induced pluripotency stem cells (hIPSCs). From a broad perspective our approach will have a significant impact in the stem cell field as it cross validates studies in hIPSCs for the study of neurodegenerative diseases. By conducting parallel analysis of hESC and hIPSC lines we will also address the genetic heterogeneity of NPC, and confirm that pathologic phenotypes found in these cells are specifically due to lack of normal NPC1 function. We will use shRNA mediated silencing of NPC1 and insertional methods of viral reprogramming to generate independent sets of NPC1 knockdown hESC lines, and hIPSC lines reprogrammed from NPC fibroblasts respectively. We will follow strict criteria of characterization of these newly generated lines to ensure they maintain stem cell properties, are genetically stable and replicate basic NPC phenotypes described in the mouse literature. We will use protocols we have developed in my lab to generate populations of pure human neurons that we will study in bulk, pure, and compartmented cultures. We will analyze human neurons derived from NPC hESCs and hIPSCs to test specific predictions that have never been probed in a human neuronal model of the disease;i) we will confirm and expand phenotypes typical of NPC that have not been tested in live human neurons, ii) we will evaluate the viability and differentiation capacity of wild type and NPC1 neurons, iii) we will measure the kinetics of cholesterol and lysosomal trafficking and the role of cholesterol in neuronal growth and survival, and iv) we will explore whether NPC is a cell autonomous disease or if neuronal failure in NPC can be affected by the glial environment. Insights generated from our observations have the potential to drastically increase our understanding of how NPC1 causes neuronal failure in humans and guide efforts that may lead to the development of a cure. UCSD is one of the country's leading research institutions. According to data from the U.S. National Science Foundation, UCSD expended nearly $800 million for research and development during the 2007 fiscal year. This project will add to our institution as an economic and academic engine at the state and national level. Our project will contribute to the continued development of the regional and national economy through research, innovation and job creation. Future economic security will be promoted through the continued employment of a senior physician scientist, a senior research technician, as well as the employment of graduate and undergraduate students. The long term impact of this investment will be the creation of knowledge, the preparation of the next generation of academic workforce, the development of new technologies, opportunities for future research and the use of commercial services in the academic sector that have the potential to yield more jobs. PUBLIC HEALTH RELEVANCE: We propose to create the first human neuronal model of Niemann Pick type C1 from human embryonic stem cells and human induced pluripotent stem cells. We will use control and NPC1 human neurons to study the kinetics of cholesterol trafficking in NPC1 and the role of cholesterol in neuronal survival and regeneration. This work will shed important light on an important childhood neurodegenerative disease and on Alzheimer's Disease as well.